Trigeminal afferents containing glutamate and/or substance P (SP) convey noxious input from orofacial regions to the dorsal horn of trigeminal nucleus caudalis (Vc). The postsynaptic effects of glutamate released from these terminals are partially mediated through the NMDA receptor and can be inhibited by ligands of the mu opiate receptor (muOR). Three specific aims of this proposal will examine the cellular substrates for potential function of the NMDA and muORs in the rat trigeminal dorsal horn using electron microscopic immunocytochemistry. Aim 1a will test the hypothesis that NMDA receptors are located within neurons postsynaptic to SP terminals, supporting the notion that agonists of the NMDA receptor facilitate the postsynaptic effects of SP on second-order neurons. Aim 1b will determine if muORs are contained within SP terminals, which would imply that the antinociceptive effects of muOR ligands can be attributed to direct modulation of SP release rather than to actions on interneurons. Aim 2 will examine the localization of NMDA receptors and muORs relative to trigeminothalamic and trigeminoparabrachial neurons that are known to be critical for the perception of head pain. These studies will use retrograde tracing from selected regions combined with immunocytochemical receptor localization. These experiments will test the hypotheses that: (a) NMDA receptors located on the plasma membranes of trigeminothalamic neurons are a substrate for glutamatergic excitation of these neurons; and (b) muORs on these cells are a potential substrate for antinociception. Preferential localization of receptors on cells projecting to thalamus may suggest models for targeted modulation of nociceptive transmission. Aim 3 will compare the subcellular localization of these receptors (NMDA and muOR) in normal and morphine tolerant rats. The muOR is critical for both the analgesia and tolerance produced by morphine and antagonists of the NMDA receptor can block morphine tolerance. These studies will determine if there is a change in receptor density and/or subcellular redistribution (e.g. shift of receptor from membrane to intracellular sites) that may be a mechanism for morphine tolerance. The experiments outlined in this proposal will demonstrate the subcellular localization of NMDA receptors and muORs in trigeminal nociceptive pathways which may be used as targets for new therapeutic strategies to control trigeminal pain, including tooth pain and headache.